The present invention relates to substituted phenylcyclohexanecarboxamides, to a process for their preparation and to their use in medicaments, in particular for the prevention and/or treatment of cardiovascular disorders, for example for the acute and chronic treatment of ischaemic disorders.
Adenosine is an endogenic effector with cell-protective activity, in particular under cell-damaging conditions with limited oxygen supply, such as, for example, in the case of ischaemia. Adenosine is a highly effective vasodilator. It increases ischaemic xe2x80x9cpreconditioningxe2x80x9d (R. Strasser, A. Vogt, W. Scharper, Z. Kardiologie 85, 1996, 79-89) and can promote the growth of collateral vessels. It is released under hypoxic conditions, for example in the case of cardiac or peripheral occlusive diseases (W. Makarewicz xe2x80x9cPurine and Pyrimidine Metabolism in Manxe2x80x9d, Plenum Press New York, 11, 1998, 351-357). Accordingly, adenosine protects against the effects of disorders caused by ischaemia, for example by increasing the coronary or peripheral circulation by vasodilation, by inhibiting platelet aggregation and by stimulating angiogenesis. Compared to systemically administered adenosine, the adenosine-uptake inhibitors have the advantage of selectivity for ischaemia. Moreover, systemically administered adenosine has a very short half-life. Systemically administered adenosine causes a strong systemic lowering of the blood pressure, which is undesirable, since circulation into the ischaemic regions may be reduced even further (xe2x80x9csteal phenomenonxe2x80x9d, L. C. Becker, Circulation 57, 1978, 1103-1110). The adenosine-uptake inhibitor increases the effect of the adenosine which is formed locally owing to the ischaemia and thus only dilates the vessels in the ischaemic regions. Accordingly, orally or intravenously administered adenosine-uptake inhibitors can be used for preventing and/or treating ischaemic disorders.
Furthermore, there have been various indications of a neuroprotective, anticonvulsive, analgesic and sleep-inducing potential of adenosine-uptake inhibitors, since they increase the intrinsic effects of adenosine by inhibiting its cellulare re-uptake (K. A. Rudolphi et al., Cerebrovascular and Brain Metabolism Reviews 4, 1992, 364-369; T. F. Murray et al., Drug Dev. Res. 28, 1993, 410-415; T. Porkka-Heiskanen et al., Science 276, 1997, 1265-1268; xe2x80x98Adenosine in the Nervous Systemxe2x80x99, Ed.: Trevor Stone, Academic Press Ltd. 1991, 217-227; M. P. DeNinno, Annual Reports in Medicinal Chemistry 33, 1998, 111-120).
It is an object of the present invention to provide novel substances for preventing and/or treating cardiovascular disorders, the substances having improved administration properties.
The present invention relates to compounds of the formula 
in which
D represents a radical 
in which
R2 represents hydrogen, halogen, hydroxyl, carboxyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C1-C6)-alkyl, (C1-C6)-alkoxy or (C1-C6)-alkoxycarbonyl,
A represents an oxygen atom or a group of the formula Nxe2x80x94R5 or CHxe2x80x94R6,
in which
R5 represents hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl, where alkyl and cycloalkyl for their part may be substituted up to three times independently of one another by hydroxyl or mono- or di-(C1-C6)-alkylamino, represents (C6-C10)-aryl, 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and S or 5- or 6-membered heterocyclyl having up to three heteroatoms from the group consisting of N, O and S, where aryl, heteroaryl and heterocyclyl for their part may be substituted up to three times independently of one another by halogen, hydroxyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C1-C6)-alkyl, (C1-C6)-alkoxy, (C1-C6)-alkoxycarbonyl or mono- or di-(C1-C6)-alkylamino,
R6 represents hydrogen, (C1-C6)-alkoxycarbonyl or carboxyl,
R1 represents hydrogen, (C1-C6)-alkyl, which for its part may be substituted by hydroxyl or (C1-C4)-alkoxy, represents (C3-C7)-cycloalkyl, (C6-C10)-aryl, 5- to 10-membered heteroaryl having up to two heteroatoms from the group consisting of N, O and S, where aryl and heteroaryl for their part may be substituted independently of one another by halogen, or represents a radical of the formula xe2x80x94NR7R8 or xe2x80x94OR9,
in which
R7 and R8 independently of one another represent hydrogen, (C6-C10)-aryl, adamantyl, (C1-C8)-alkyl, whose chain may be interrupted by one or two oxygen atoms and which may be substituted up to three times independently of one another by hydroxyl, phenyl, trifluoromethyl, (C3-C8)-cycloalkyl, (C1-C6)-alkoxy, mono- or di-(C1-C6)-alkylamino, 5- or 6-membered heterocyclyl having up to three heteroatoms from the group consisting of N, O and S or by 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and S, represent (C3-C8)-cycloalkyl, which may be substituted up to three times independently of one another by (C1-C4)-alkyl, hydroxyl or oxo, or represent 5- or 6-membered heterocyclyl having up to two heteroatoms from the group consisting of N, O and S, where N is substituted by hydrogen or (C1-C4)-alkyl, or
R7 and R8 together with the nitrogen atom to which they are attached form a 4- to 7-membered saturated heterocycle which may contain up to two further heteroatoms from the group consisting of N, O and S and which is optionally substituted by hydroxyl, oxo or (C1-C6)-alkyl, which for its part may be substituted by hydroxyl, and
R9 represents (C6-C10)-aryl, adamantyl, (C1-C8)-alkyl, whose chain may be interrupted by one or two oxygen atoms and which may be substituted up to three times independently of one another by hydroxyl, phenyl, trifluoromethyl, (C3-C8)-cycloalkyl, (C1-C6)-alkoxy, mono- or di-(C1-C6)-alkylamino, 5- or 6-membered heterocyclyl having up to three heteroatoms from the group consisting of N, O and S or by 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and S, represents (C3-C8)-cycloalkyl, which may be substituted up to three times independently of one another by (C1-C4)-alkyl, hydroxyl or oxo, or represents 5- or 6-membered heterocyclyl having up to two heteroatoms from the group consisting of N, O and S, where N is substituted by hydrogen or (C1-C4)-alkyl,
R3 represents (C1-C8)-alkyl, whose chain may be interrupted by a sulphur or oxygen atom or an S(O) or SO2 group, represents phenyl, benzyl or 5- or 6-membered heteroaryl having up to two heteroatoms from the group consisting of N, O and S, where phenyl, benzyl and heteroaryl may be substituted up to three times independently of one another by halogen, trifluoromethyl, cyano, nitro, hydroxyl, (C1-C6)-alkyl or (C1-C6)-alkoxy, and
R4 represents a radical of the formula xe2x80x94C(O)xe2x80x94NR10R11,
in which
R10 and R11 independently of one another represent hydrogen or (C1-C6)-alkyl,
and their salts, hydrates, hydrates of the salts and solvates.
Salts of the compounds according to the invention are physiologically acceptable salts of the substances according to the invention with mineral acids, carboxylic acids or sulphonic acids. Particular preference is given, for example, to salts of hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonic acid, toluenesulphonic acid, benzenesulphonic acid, naphthalenedisulphonic acids, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.
Salts can also be physiologically acceptable metal or ammonium salts of the compounds according to the invention. Particularly preferred are alkali metal salts (for example sodium salts or potassium salts), alkaline earth metal salts (for example magnesium salts or calcium salts), and also ammonium salts, which are derived from ammoia or organic amines, such as, for example, ethylamine, di- or triethylamine, di- or triethanolamine, dicyclohexylamine, dimethylaminoethanol, arginine, lysine, ethylenediamine or 2-phenylethylamine.
Depending on the substitution pattern, the compounds according to the invention can exist in stereoisomeric forms which are either like image and mirror image (enantiomers) or which are not like image and mirror image (diastereomers). The invention relates both to the enantiomers or diastereomers and to their respective mixtures. The racemic forms, like the diastereomers, can be separated in a known manner into the stereoisomerically uniform components.
Moreover, the invention also includes prodrugs of the compounds according to the invention. According to the invention, prodrugs are those forms of the compounds of the above formula (I) which for their part may be biologically active or inactive, but which are converted under physiological conditions (for example metabolically or solvolytically) into the corresponding biologically active form.
According to the invention, xe2x80x9chydratesxe2x80x9d or xe2x80x9csolvatesxe2x80x9d are those forms of the compounds of the above formula (I) which, in solid or liquid state, form a molecular compound or a complex by hydration with water or coordination with solvent molecules. Examples of hydrates are sesquihydrates, monohydrates, dihydrates and trihydrates. Equally suitable are the hydrates or solvates of salts of the compounds according to the invention.
Halogen represents fluorine, chlorine, bromine and iodine. Preference is given to chlorine or fluorine.
(C1-C8)-alkyl represents a straight-chain or branched alkyl radical having 1 to 8 carbon atoms. Examples which may be mentioned are: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl and n-octyl. The corresponding alkyl groups having fewer carbon atoms, such as, for example (C1-C6)-alkyl, (C1-C4)-alkyl and (C1-C3)-alkyl, are derived analogously from this definition. In general, (C1-C3)-alkyl is preferred.
The meaning of the corresponding component of other, more complex substituents, such as, for example, di-alkylamino, mono- or di-alkylamino is also derived from this definition.
Mono- or di-(C1-C4)-alkylamino represents an amino group having one or two identical or different straight-chain or branched alkyl substituents of in each case 1 to 4 carbon atoms. Examples which may be mentioned are: methylamino, ethylamino, n-propylamino, isopropylamino, tert-butylamino, N,N-dimethylamino, N,N-diethylamino, N-ethyl-N-methylamino, N-methyl-N-n-propylamino, N-isopropyl-N-n-propylamino and N-t-butyl-N-methyl amino.
(C3-C8)-Cycloalkyl represents a cyclic alkyl radical having 3 to 8 carbon atoms. Examples which may be mentioned are: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. The corresponding cycloalkyl groups having fewer carbon atoms, such as, for example, (C3-C7)-cycloalkyl or (C3-C6)-cycloalky, are derived analogously from this definition. Preference is given to cyclopropyl, cyclopentyl and cyclohexyl.
(C1-C6)-Alkoxy represents a straight-chain or branched alkoxy radical having 1 to 6 carbon atoms. Examples which may be mentioned: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentoxy and n-hexoxy. The corresponding alkoxy groups having fewer carbon atoms, such as, for example, (C1-C4)-alkoxy or (C1-C3)-alkoxy, are derived analogously from this definition. In general, (C1-C3)-alkoxy is preferred.
The meaning of the corresponding component of other, more complex substituents, such as, for example, alkoxy carbonyl, which represents an alkoxy radical which is attached via a carbonyl group, is also derived from this definition.
(C6-C10)-Aryl represents an aromatic radical having 6 to 10 carbon atoms. Examples which may be mentioned are: phenyl and naphthyl.
5- to 10-membered heteroaryl having up to 3 heteroatoms from the group consisting of N, O and S represents a mono- or bicyclic heteroaromatic which is attached via a ring carbon atom of the heteroaromatic, if appropriate also via a ring nitrogen atom of the heteroaromatic. Examples which may be mentioned are: pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, thienyl, furyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, thiazolyl, oxazolyl, oxdiazolyl, isoxazolyl, benzofuranyl, benzothienyl or benzimidazolyl. The corresponding heterocycles having fewer heteroatoms, such as, for example, those having up to 2 heteroatoms from the group consisting of N, O and S are derived analogously from this definition. In general, preference is given to 5- or 6-membered aromatic heterocycles having up to 2 heteroatoms from the group consisting of N, O and S, such as, for example, pyridyl, pyrimidyl, pyridazinyl, furyl, imidazolyl and thienyl.
5- or 6-membered heterocyclyl having up to 3 heteroatoms from the group consisting of N, O and S represents a saturated or partially unsaturated heterocycle which is attached via a ring carbon atom or a ring nitrogen atom. Examples which may be mentioned are: tetrahydrofuryl, pyrrolidinyl, pyrrolinyl, dihydropyridinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl. Preference is given to saturated heterocycles, in particular to piperidinyl, piperazinyl, morpholinyl and pyrrolidinyl.
The compounds of the formula (I) according to the invention can be present in at least eight different configurations, the four different configurations (Ia) to (Id) below being preferred: 
Particular preference is given to the configuration (Id).
Preference is furthermore given to compounds of the formula (I) according to the invention
in which
D represents a radical 
in which
R2 represents hydrogen, halogen, hydroxyl, carboxyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C1-C6)-alkyl, (C1-C6)-alkoxy or (C1-C6)-alkoxycarbonyl,
A represents an oxygen atom or a group of the formula Nxe2x80x94R5 or CHxe2x80x94R6,
in which
R5 represents hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl, where alkyl and cycloalkyl for their part may be substituted up to three times independently of one another by hydroxyl or mono- or di-(C1-C6)-alkylamino, represents (C6-C10)-aryl, 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and S or 5- or 6-membered heterocyclyl having up to three heteroatoms from the group consisting of N, O and S, where aryl, heteroaryl and heterocyclyl for their part may be substituted up to three times independently of one another by halogen, hydroxyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C1-C6)-alkyl, (C1-C6)-alkoxy, (C1-C6)-alkoxycarbonyl or mono- or di-(C1-C6)-alkylamino,
R6 represents hydrogen, (C1-C6)-alkoxycarbonyl or carboxyl,
R1 represents hydrogen, (C1-C6)-alkyl, which for its part may be substituted by hydroxyl or (C1-C4)-alkoxy, represents (C3-C7)-cycloalkyl, (C6-C10)-aryl, 5- to 10-membered heteroaryl having up to two heteroatoms from the group consisting of N, O and S, where aryl and heteroaryl for their part may be substituted independently of one another by halogen, or represents a radical of the formula xe2x80x94NR7R8 or xe2x80x94OR9,
in which
R7 and R8 independently of one another represent hydrogen, (C6-C10)-aryl, adamantyl, (C1-C8)-alkyl, whose chain may be interrupted by one or two oxygen atoms and which may be substituted up to three times independently of one another by hydroxyl, phenyl, trifluoromethyl, (C3-C8)-cycloalkyl, (C1-C6)-alkoxy, mono- or di-(C1-C6)-alkylamino, 5- or 6-membered heterocyclyl having up to three heteroatoms from the group consisting of N, O and S or by 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and S, represent (C3-C8)-cycloalkyl, which may be substituted up to three times independently of one another by (C1-C4)-alkyl, hydroxyl or oxo, or represent 5- or 6-membered heterocyclyl having up to two heteroatoms from the group consisting of N, O and S, where N is substituted by hydrogen or (C1-C4)-alkyl,
R7 and R8 together with the nitrogen atom to which they are attached form a 4- to 7-membered saturated heterocycle which may contain up to two further heteroatoms from the group consisting of N, O and S and which is optionally substituted by hydroxyl, oxo or (C1-C6)-alkyl, which for its part may be substituted by hydroxyl, and
R9 represents (C6-C10)-aryl, adamantyl, (C1-C8)-alkyl, whose chain may be interrupted by one or two oxygen atoms and which may be substituted up to three times independently of one another by hydroxyl, phenyl, trifluoromethyl, (C3-C8)-cycloalkyl, (C1-C6)-alkoxy, mono- or di-(C1-C6)-alkylamino, 5- or 6-membered heterocyclyl having up to three heteroatoms from the group consisting of N, O and S or by 5- to 10-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and S, represents (C3-C8)-cycloalkyl, which may be substituted up to three times independently of one another by (C1-C4)-alkyl, hydroxyl or oxo, or represents 5- or 6-membered heterocyclyl having up to two heteroatoms from the group consisting of N, O and S, where N is substituted by hydrogen or (C1-C4)-alkyl,
R3 represents (C1-C8)-alkyl, whose chain may be interrupted by a sulphur atom or an S(O) or SO2 group, represents phenyl, benzyl or 5- or 6-membered heteroaryl having up to two heteroatoms from the group consisting of N, O and S, where phenyl, benzyl and heteroaryl may be substituted up to three times independently of one another by halogen, trifluoromethyl, cyano, nitro, hydroxyl, (C1-C6)-alkyl or (C1-C6)-alkoxy, and
R4 represents a radical of the formula xe2x80x94C(O)xe2x80x94NR10R11,
in which
R10 and R11 independently of one another represent hydrogen or (C1-C6)-alkyl,
and their salts, hydrates, hydrates of the salts and solvates.
Particular preference is given to compounds of the formula (I) according to the invention,
in which
D represents a radical 
in which
R2 represents hydrogen, chlorine or fluorine,
A represents an oxygen atom or a group of the formula Nxe2x80x94R5,
in which
R5 represents hydrogen, (C1-C6)-alkyl, which for its part may be substituted up to two times by hydroxyl, represents (C3-C7)-cycloalkyl, phenyl or 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and S, where phenyl and heteroaryl for their part may be substituted up to two times independently of one another by halogen, cyano, trifluoromethyl, trifluoromethoxy, (C1-C4)-alkyl, (C1-C4)-alkoxy or di-(C1-C4)-alkylamino,
R1 represents hydrogen, (C1-C6)-alkyl, which for its part may be substituted by hydroxyl or (C1-C4)-alkoxy, represents (C3-C7)-cycloalkyl, phenyl, 5- or 6-membered heteroaryl having up to two heteroatoms from the group consisting of N, O and S, where phenyl and heteroaryl for their part independently of one another may be substituted by halogen, or represents a radical of the formula xe2x80x94NR7R8 or xe2x80x94OR9,
in which
R7 and R8 independently of one another represent hydrogen, phenyl, adamantyl, (C1-C6)-alkyl, whose chain may be interrupted by one or two oxygen atoms and which may be substituted up to two times independently of one another by hydroxyl, phenyl, trifluoromethyl, (C3-C6)-cycloalkyl, (C1-C4)-alkoxy, mono- or di-(C1-C4)-alkylamino, 5- or 6-membered heterocyclyl having up to two heteroatoms from the group consisting of N and O or by 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and S, represents (C3-C8)-cycloalkyl, which may be substituted up to two times by hydroxyl, or represent 5- or 6-membered heterocyclyl having up to two heteroatoms from the group consisting of N, O and S, where N is substituted by hydrogen or (C1-C4)-alkyl, or
R7 and R8 together with the nitrogen atom to which they are attached form a 4- to 7-membered saturated heterocycle which may contain up to two further heteroatoms from the group consisting of N, O and S and which is optionally substituted by hydroxyl, oxo or (C1-C6)-alkyl, which for its part may be substituted by hydroxyl, and
R9 represents phenyl, adamantyl, (C1-C6)-alkyl, whose chain may be interrupted by one or two oxygen atoms and which may be substituted up to two times independently of one another by hydroxyl, phenyl, trifluoromethyl, (C3-C6)-cycloalkyl, (C1-C3)-alkoxy, mono- or di-(C1-C4)-alkylamino, 5- or 6-membered heterocyclyl having up to two heteroatoms from the group consisting of N and O or by 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and S, represents (C3-C8)-cycloalkyl, which may be substituted up to two times by hydroxyl, or represents 5- or 6-membered heterocyclyl having up to two heteroatoms from the group consisting of N, O and S, where N is substituted by hydrogen or (C1-C4)-alkyl,
R3 represents (C1-C8)-alkyl, whose chain may be interrupted by a sulphur atom or an S(O) or SO2 group, represents phenyl, benzyl or 5- or 6-membered heteroaryl having up to two heteroatoms from the group consisting of N, and S, where phenyl, benzyl and heteroaryl may be substituted up to two times independently of one another by halogen, trifluoromethyl, cyano, (C1-C3)-alkyl, (C1-C3)-alkoxy or hydroxyl, and
R4 represents a radical of the formula xe2x80x94C(O)xe2x80x94NR10R11,
in which
R10 and R11 independently of one another represent hydrogen or (C1-C6)-alkyl,
and their salts, hydrates, hydrates of the salts and solvates.
Very particular preference is given to compounds of the formula (I) according to the invention
in which
D represents a radical 
in which
R2 represents hydrogen,
A represents an oxygen atom or a group of the formula Nxe2x80x94R5,
in which
R5 represents hydrogen, (C1-C6)-alkyl, which for its part may be substituted up to two times by hydroxyl, represents (C3-C7)-cycloalkyl, phenyl or 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and S, where phenyl and heteroaryl for their part may be substituted up to two times independently of one another by fluorine, chlorine, cyano, trifluoromethyl, trifluoromethoxy, (C1-C3)-alkyl, (C1-C3)-alkoxy or di-(C1-C3)-alkylamino,
R1 represents (C1-C4)-alkyl or a radical of the formula xe2x80x94NR7R8,
in which
R7 and R8 independently of one another represent hydrogen, phenyl, adamantyl, (C1-C4)-alkyl, whose chain may be interrupted by one or two oxygen atoms and which may be substituted up to two times independently of one another by hydroxyl, phenyl, trifluoromethyl, (C3-C6)-cycloalkyl, (C1-C3)-alkoxy, mono- or di-(C1-C3)-alkylamino, 5- or 6-membered heterocyclyl having up to two heteroatoms from the group consisting of N and O or by 5- or 6-membered heteroaryl having up to three heteroatoms from the group consisting of N, O and S, represent (C3-C8)-cycloalkyl, which may be substituted up to two times by hydroxyl, or represents 5- or 6-membered heterocyclyl having up to two heteroatoms from the group consisting of N, O and S, where N is substituted by hydrogen or (C1-C4)-alkyl, or R7 and R8 together with the nitrogen atom to which they are attached form a 4- to 7-membered saturated heterocycle which may contain up to two further heteroatoms from the group consisting of N, O and S and which is optionally substituted by by hydroxyl, oxo or (C1-C6)-alkyl, which for its part may be substituted by hydroxyl, R3 represents (C1-C8)-alkyl, whose chain may be interrupted by a sulphur atom or an S(O) or SO2 group, represents phenyl, benzyl or 5- or 6-membered heteroaryl having up to two heteroatoms from the group consisting of N, O and S, where phenyl, benzyl and heteroaryl may be substituted up to two times independently of one another by halogen, trifluoromethyl, cyano, (C1-C3)-alkyl, (C1-C3)-alkoxy or hydroxyl, and
R4 represents a radical of the formula xe2x80x94C(O)xe2x80x94NR10OR11,
in which
R10 and R11 independently of one another represent hydrogen, methyl or ethyl,
and their salts, hydrates, hydrates of the salts and solvates.
Most particular preference is given to compounds of the formula (I),
in which
D is a radical 
in which
R2 represents hydrogen,
A represents an oxygen atom or a group of the formula Nxe2x80x94R5,
in which
R5 represents (C3-C7)-cycloalkyl, phenyl, which for its part may be substituted by fluorine, or represents pyridyl,
R1 represents methyl or a radical of the formula xe2x80x94NR7R8,
in which
R7 and R8 independently of one another represent (C1-C4)-alkyl, which may be mono- or disubstituted by hydroxyl, or
R7 and R8 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated heterocycle which may contain a further heteroatom O or N, where N is substituted by hydrogen or (C1-C3)-alkyl, which for its part may be substituted by hydroxyl,
R3 represents phenyl, which is optionally substituted in the para-position by fluorine, or represents pyridyl, and
R4 represents a radical of the formula xe2x80x94C(O)xe2x80x94NR10R11,
in which
R10 and R11 represent hydrogen,
and their salts, hydrates, hydrates of the salts and solvates.
Most particular preference is also given to:
(1R,2R)-N-[(1S)-2-amino-1-(4-fluorophenyl)-2-oxoethyl]-2-(4-{[{[ethyl(2-hydroxyethyl)amino]carbonyl}(4-fluorophenyl)amino]methyl}phenyl)cyclohexanecarboxamide 
(1R,2R)-N-[(1S)-2-amino-2-oxo-1-phenylethyl]-2-(4-{[[(dimethylamino)carbonyl]-(phenyl)amino]methyl}phenyl)cyclohexanecarboxamide 
(1R,2R)-N-[(1S)-2-amino-2-oxo-1-phenylethyl]-2-[4-({cyclopropyl[(dimethylamino)carbonyl]amino}methyl)phenyl]cyclohexanecarboxamide 
(1R,2R)-N-[(1S)-2-amino-2-oxo-1-phenylethyl]-2-(4-{[[(diethylamino)carbonyl](2-pyridinyl)amino]methyl}phenyl)cyclohexanecarboxamide 
N-{4-[(1R,2R)-2-({[(1S)-2-amino-2-oxo-1-phenylethyl]amino}carbonyl)cyclohexyl]-benzyl}-N-phenyl-4-morpholinecarboxamide 
(S)-N-{{(1R,2R)-2-(4-{[{[2-hydroxylethylamino]carbonyl}(phenyl)amino]methyl}phenyl)cyclohex-1-yl}carbonyl}-phenylglycinamide 
(1R,2R)-2-(4-{[acetyl(2-pyridinyl)amino]methyl}phenyl)-N-[(1S)-2-amino-2-oxo-1-phenylethyl]cyclohexanecarboxamide 
(1R,2R)-N-[(1S)-2-amino-1-phenyl-2-oxoethyl]-2-(4-{[{[ethyl(2-hydroxyethyl)amino]carbonyl}(phenyl)amino]methyl}phenyl)cyclohexanecarboxamide 
4-[(1R,2R)-2-(({[(1S)-2-amino-1-(4-fluorophenyl)-2-oxoethyl]amino}carbonyl)cyclohexyl]benzyl 4-(2-hydroxyethyl)-1-piperazinecarbamate 
4-[(1R,2R)-2-({[(1S)-2-amino-1-phenyl-2-oxoethyl]amino}carbonyl)cyclohexyl]benzyl-4-(2-hydroxyethyl)-1-piperazinecarbamate 
and their salts, hydrates, hydrates of the salts and solvates.
Moreover, we have found a process for preparing the compounds of the formula (I) according to the invention where
[A] compounds of the formula (II) 
in which
D is as defined above,
T represents (C1-C4)-alkyl, preferably methyl or tert-butyl, and
V represents a suitable leaving group, such as, for example, halogen, mesylate or tosylate, preferably bromine,
are initially converted by reaction with compounds of the formula (III)
Bxe2x80x94Hxe2x80x83xe2x80x83(III), 
in which
B represents 
or
optionally, if R1 reprsents OR9, represents 
and
R1 and A are each as defined above, where any amino and hydroxyl functions which may be present are optionally blocked by customary amino or hydroxyl protective groups,
and to the compounds of the formula (IV) 
in which B, D and T are each as defined above,
these reaction mixtures obtained are in a next step converted with acids or bases into the corresponding carboxylic acids of the formula (V) 
in which
R1, A and D are as defined above,
which are, if appropriate, activated, in particular by conversion into a corresponding carboxylic acid derivative, such as a carbonyl halide, a carboxylic anhydride or a carboxylic acid,
and these compounds are finally reacted in inert solvents according to known methods with compounds of the formula (VI) or salts thereof 
in which
R3 and R4 are as defined above, or
[B] if A represents an oxygen atom or NR5,
compounds of the formula (VII) 
in which
D, R3 and R4 are as defined above
and
A represents an oxygen atom or a group of the formula Nxe2x80x94R5,
where R5 is as defined above,
if appropriate in the presence of a base,
are reacted either with compounds of the formula (VIII) 
in which
R1 is as defined above and W represents a suitable leaving group, such as, for example, the corresponding symmetric anhydride or a halogen, preferably chlorine, or
with a phosgene equivalent, such as, for example, disuccinimidyl carbonate, and then with compounds of the formula (IX)
R7R8NHxe2x80x83xe2x80x83(IX), 
in which
R7 and R8 are as defined above, or
with an isocyanate of the formula (X)
R7NCOxe2x80x83xe2x80x83(X), 
in which
R7 is as defined above.
The compounds of the formula (I) obtained according to process variant [A] or [B] can, if appropriate, subsequently be converted into the corresponding salts, for example by reaction with an acid.
The compounds of the corresponding diastereomeric and enantiomeric forms are prepared correspondingly, either using enantiomerically or diastereomerically pure starting materials or by subsequent separation of the racemates formed by customary methods (for example racemate resolution, chromatography on chiral columns, etc.).
The process according to the invention is illustrated in an exemplary manner by the equation below: 
If R1 represents OR9, the following synthesis sequence is likewise possible: 
The process according to the invention is generally carried out under atmospheric pressure. However, it is also possible to carry out the process under elevated pressure or under reduced pressure (for example in a range from 0.5 to 5 bar).
In the context of the invention, customary amino protective groups are the amino protective groups used in peptide chemistry.
These preferably include: benzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, allyloxycarbonyl, vinyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, cyclohexoxycarbonyl, 1,1-dimethylethoxycarbonyl, adamantylcarbonyl, phthaloyl, 2,2,2-trichlorethoxycarbonyl, 2,2,2-trichloro-tert-butoxycarbonyl, Menthyloxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, formyl, acetyl, propionyl, pivaloyl, 2-chloroacetyl, 2-bromoacetyl, 2,2,2-trifluoroacetyl, 2,2,2-trichloroacetyl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, phthalimido, isovaleroyl or benzyloxymethylene, 4-nitrobenzyl, 2,4-dinitrobenzyl or 4-nitrophenyl. A preferred protective group for primary amines is phthalimide. Preferred protective groups for secondary amines are benzyloxycarbonyl and tert-butoxycarbonyl.
The amino protective groups are removed in a manner known per se, using, for example, hydrogenolytic, acidic or basic conditions, preferably acids, such as, for example, hydrochloric acid or trifluoroacetic acid, in inert solvents, such as ether, dioxane and methylene chloride.
In the context of the definition given above, a customary hydroxyl protective group is generally a protective group from the group: trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyl-dimethylsilyl, tert-butyldiphenylsilyl, dimethylhexylsilyl, dimethylthexylsilyl, trimethylsilylethoxycarbonyl, benzyl, triphenylmethyl(trityl), monomethoxytrityl (MMTr), dimethyloxytrityl (DMTr), benzyloxycarbonyl, 2-nitrobenzyl, 4-nitrobenzyl, 2-nitrobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, tert-butyloxycarbonyl, 4-methoxybenzyl, 4-methoxybenzyloxycarbonyl, formyl, acetyl, trichloroacetyl, 2,2,2-trichloroethoxycarbonyl, 2,4-dimethoxybenzyl, 2,4-dimethoxybenzyloxycarbonyl, methoxymethyl, methylthiomethyl, methoxyethoxymethyl, [2-(trimethylsilyl)ethoxy]-methyl, 2-(methylthiomethoxy)ethoxycarbonyl, tetrahydropyranyl, benzoyl, N-succinimide, 4-methylbenzoyl, 4-nitrobenzoyl, 4-fluorobenzoyl, 4-chlorobenzoyl or 4-methoxybenzoyl. Preference is given to tert-butyl-dimethylsilyl.
The hydroxyl protective group is removed in a manner known per se, for example using acid, base or by addition of tetrabutylammonium fluoride.
Solvents suitable for the process are customary organic solvents which do not change under the reaction conditions. These include ethers, such as diethyl ether, dioxane, tetrahydrofuran, glycol dimethyl ether, or hydrocarbons, such as benzene, toluene, xylene, hexane, cyclohexane or mineral oil fractions, or halogenated hydrocarbons, such as dichloromethane, trichloromethane, carbon tetrachloride, dichloroethylene, trichloroethylene or chlorbenzene, or ethyl acetate, pyridine, dimethyl sulphoxide, dimethylformamide, N,Nxe2x80x2-dimethylpropyleneurea (DMPU), N-methylpyrrolidone (NMP), acetonitrile, acetone or nitromethane. It is also possible to use mixtures of the solvents mentioned.
Bases suitable for the process according to the invention are, in general, inorganic or organic bases. These preferably include alkali metal hydroxides, such as, for example, sodium hydroxide or potassium hydroxide, alkaline earth metal hydroxides, such as, for example, barium hydroxide, alkali metal carbonates, such as sodium carbonates, potassium carbonate or caesium carbonate, alkaline earth metal carbonate, such as calcium carbonate, or alkali metal or alkaline earth metal alkoxides, such as sodium methoxide or potassium methoxide, sodium ethoxide or potassium ethoxide or potassium tert-butoxide, or organic amines, such as triethylamine, or heterocycles, such as 1,4-diazabicyclo[2.2.2]octane (DABCO), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), pyridine, diaminopyridine, N-methylpiperidine or N-methyl-morpholine. It is also possible to use alkali metals such as sodium or their hydrides, such as sodium hydride, as bases.
Preferred solvents for process step [A] (II)+(m)xe2x86x92(IV) are diethyl ether, tetrahydrofuran and dimethylformamide. Particular preference is given to di methyl formamide.
Preferred bases for process step [A] (II)+(III)xe2x86x92(IV) are sodium hydride and sodium hydroxide.
In general, the base is employed in an amount of from 0.05 mol to 10 mol, preferably from 1 mol to 2 mol, based on 1 mol of the compound of the formula (II).
The process step [A] according to the invention, (II)+(III)xe2x86x92(IV), is generally carried out in a temperature range of from xe2x88x9220xc2x0 C. to +100C, in particular from xe2x88x9220xc2x0 C. to +80xc2x0 C., preferably from 0xc2x0 C. to +80xc2x0 C.
The hydrolysis of carboxylic esters in process step [A] (W)xe2x86x92(V) is carried out by customary methods by treating the esters in inert solvents with bases, and converting the salts which are initially formed into the free carboxylic acids, by treatment with acid. In the case of the tert-butyl esters, the hydrolysis is preferably carried out using acids.
Solvents suitable for the hydrolysis of the carboxylic esters are water or the organic solvents which are customary for ester hydrolysis. These preferably include alcohols, such as methanol, ethanol, propanol, isopropanol or butanol, or ethers, such as tetrahydrofuran or dioxane, dimethylformamide, dichloromethane or dimethyl sulphoxide. It is also possible to use mixtures of the solvents mentioned. Preference is given to water/tetrahydrofuran and, in the case of the reaction with trifluoroacetic acid, dichlormethane and, in the case of hydrogen chloride, tetrahydrofuran, diethyl ether, dioxane or water.
Suitable bases are the inorganic bases customary for hydrolysis. These preferably include alkali metal hydroxides or alkaline earth metal hydroxides, such as, for example, sodium hydroxide, lithium hydroxide, potassium hydroxide or barium hydroxide, or alkali metal carbonates, such as sodium carbonate or potassium carbonate, or sodium bicarbonate. Particular preference is given to using sodium hydroxide or lithium hydroxide.
Suitable acids are, in general, trifluoroacetic acid, sulphuric acid, hydrogen chloride, hydrogen bromide and acetic acid, or mixtures thereof, if appropriate with addition of water. Preference is given to hydrogen chloride or trifluoroacetic acid in the case of the tert-butyl esters and to hydrochloric acid in the case of the methyl esters.
When carrying out the hydrolyses, the base or the acid is generally employed in an amount of from 1 to 100 mol, preferably from 1.5 to 40 mol, based on 1 mol of ester.
The hydrolysis is generally carried out in a temperature range of from 0xc2x0 C. to +100xc2x0 C.
The amide formation in process step [A] (V)+(VI)xe2x86x92(I) is preferably carried out in the solvent dimethylformamide or dichloromethane.
Preferred auxiliaries used for the amide formation are customary condensing agents, such as carbodiimides, for example N,Nxe2x80x2-diethyl-, N,N,xe2x80x2-dipropyl-, N,Nxe2x80x2-diisopropyl-, N,Nxe2x80x2-dicyclohexylcarbodiimide, N-(3-dimethylaminopropyl)-Nxe2x80x2-ethylcarbodiimide hydrochloride (EDC), or carbonyl compounds, such as carbonyldiimidazole, or 1,2-oxazolium compounds, such as 2-ethyl-5-phenyl-1,2-oxazolium 3-sulphate or 2-tert-butyl-5-methyl-isoxazolium perchlorate, or acylamino-compounds, such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, or propanephosphonic anhydride, or isobutyl chloroformate, or bis-(2-oxo-3-oxazolidinyl)-phosphoryl chloride or benzotriazolyloxy-tri(dimethylamino)phosphonium hexafluorophosphate, or O-(benzotriazol-1-yl)-N,N,Nxe2x80x2,Nxe2x80x2-tetra-methyluronium hexafluorophosphate (HBTU), 2-(2-oxo-1-(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TPTU) or O-(7-azabenzotriazol-1-yl)-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium hexafluorophosphate (HATU), if appropriate in combination with further auxiliaries, such as 1-hydroxybenzotriazole or N-hydroxysuccinimide, and the bases used are preferably alkali metal carbonates, for example sodium carbonate or potassium carbonate, or sodium bicarbonate or potassium bicarbonate, or organic bases, such as trialkylamines, for example triethylamine, N-methylmorpholine, N-methylpiperidine or diisopropylethylamine. Particular preference is given to the combination of EDC, N-methylmorpholine and 1-hydroxybenzotriazole.
The amide formation is generally carried out a temperature range of from 0xc2x0 C. to +10xc2x0 C.
Solvents suitable for the acylation in process step [B] (VII)xe2x86x92(I) are the customary solvents which are inert under the reaction conditions; preference is given here to dimethylformamide and dichloromethane.
Suitable bases used for the acylation, if appropriate, are the customary inorganic or organic bases, preferably triethylamine.
The acylation is generally carried out in a temperature range of from 0xc2x0 C. to +100xc2x0 C.
The compounds of the formulae (II), (III), (VI), (VIII), (IX) and (X) are known or can be prepared by customary methods (cf. EP-A-0 725 061, EP-A-0 725 064, EP-A-0 581 003, EP-A-0 611 767, WO-A-00/73274).
The compounds of the formula (VII) can be prepared by converting compounds of the formula (II) with compounds of the formula (XI)
Y-A-Hxe2x80x83xe2x80x83(XI), 
in which
A represents an oxygen atom or a group of the formula Nxe2x80x94R5,
where R5 is as defined above, and
Y represents a suitable amino or hydroxyl protective group,
if appropriate in the presence of a base, into compounds of the formula (XII) 
in which
A, D, T and Y are as defined above,
in the next steps, analogously to the reaction steps described under [A], initially converting these by hydrolysis into compounds of the formula (XIII), 
in which
A, D and Y are as defined above,
then reacting with compounds of the formula (VI) to give compounds of the formula (XIV) 
in which
A, D, Y, R3 and R4 are as defined above,
and finally removing the protective group Y by customary methods.
The preparation of compounds of the formula (VII) can be illustrated in an exemplary manner by the equation below: 
The conversion (II)+(XI)xe2x86x92(XII) is carried out in the customary solvents which are inert under the reaction conditions. Preference is given to acetonitrile.
Suitable bases, which are used for this reaction, if appropriate, are the customary inorganic or organic bases.
The reaction is generally carried out in a temperature range of from 0xc2x0 C. to +100xc2x0 C.
Surprisingly, the compounds of the formula (I) according to the invention have an unforeseeable useful pharmacological activity spectrum, combined with improved administration properties.
The compounds according to the invention act as adenosine-uptake inhibitors. They can be used for preparing medicaments for the prevention and/or treatment of peripheral and cardiovascular disorders caused by ischaemia, in particular for the acute and chronic treatment of ischaemic disorders of the cardiovascular system, such as, for example, coronary heart disease, stable and unstable angina pectoris, of peripheral and arterial occlusive diseases, of thrombotic vascular occlusions, of myocardial infarction and of reperfusion damage.
Moreover, owing to their potential to increase angiogenesis, they are particularly suitable for a permanent therapy of all occlusive diseases.
In addition, the compounds according to the invention, alone or in combination with other medicaments, can be used by oral or intravenous administration for preventing and/or treating cerebral ischaemia, stroke, reperfusion damage, brain trauma, oedema, spasms, epilepsy, respiratory arrest, cardiac arrest, Reye syndrome, cerebral thrombosis, embolism, tumours, haemorrhages, encephalomyelitis, hydroencephalitis, spinal injuries, post-operative brain damage, injuries of the retina or the optical nerve following glaucoma, ischaemia, hypoxia, oedema or trauma, and also in the treatment of schizophrenia, sleep disturbances and acute and/or chronic pain and also neurodegenerative disorders, in particular for the treatment of cancer-induced pain and chronic neuropathic pain, such as, for example, in cases of diabetic neuropathy, posttherpeutic neuralgia, peripheral nerve damage, central pain (for example as a result of cerebral ischaemia) and trigeminal neuralgia and other chronic pain, such as, for example, lumbago, lower back pain or rheumatic pain.
Adenosine-uptake inhibitors like the compounds according to the invention can furthermore also be used for treating hypertension and cardiac insufficiency, myocarditis, nephritis, pancreatitis, diabetic nephropathy, oedema and for potentiating the effect of nucleobase, nucleoside or nucleotide antimetabolites in cancer chemotherapy and antiviral (for example HIV) chemotherapy.
The compounds according to the invention have an increased solubility in water and an improved bioavailability, in particular when administered orally. These advantageous properties can, if appropriate, be improved even further with the aid of formulation auxiliaries and/or by adjusting a suitable pH. Good solubility in water and high bioavailability are, as is known, advantageous properties in medicinally active compounds and formulations; thus, the compounds according to the invention are, for example, particularly suitable for oral and intravenous administration.
A Assessment of the Physiological Activity
1. Determination of the Solubility
To determine the solubility, a precipitation method was used:
10 mg of the test substance are completely dissolved in 50 xcexcl of DMSO (stock solution). 20 xcexcl of this solution are added to 2000 xcexcl of physiological saline. This solution, in turn, is shaken at 25xc2x0 C. in a Thermomixer Comfort (from Eppendorf) at 1400 rpm for 24 hours for equilibration.
The precipitated fractions of the test substance are centrifuged off using a Biofuge 15 from Heraeus at 14,000 rpm for 5 min. 1300 xcexcl of the supernatant are once more centrifuged using a Microfuge from Beckmann at 45,000 rpm=125,000 g.
10 xcexcl of this centrifugation supernatant are then diluted with 1000 xcexcl of DMSO, and this solution is measured by HPLC (Hewlett Packard 1090, method: gradient from 100% PBS buffer pH=4 to 10% buffer/90% acetonitrile over a period of 15 min, column: RP18)
Using a calibration curve, the measured peak area of the HPLC measurement is converted into the substance concentration. For the calibration curve, 20 xcexcl of the stock solution are diluted successively with DMSO such that 5 concentrations of 2.5 mg/l to 2000 mg/l result. These solutions are likewise measured by HPLC (see method above), and the peak areas are plotted as a function of the concentrations.
2. Inhibition of the Adenosine Uptake in Rabbit Erythrocytes by the Compounds According to the Invention
The capability of substances to influence the adenosine-uptake system is investigated by determining the inhibitory effect of the substances on functional adenosine uptake.
For the functional adenosine-uptake test, an erythrocyte preparation from rabbit blood is used. The blood is drawn intravenously using citrate (3 ml Monovette 9NC from Sarstedt) as anticoagulant. The blood is centrifuged at 3000 g for 5 min and the erythrocytes are suspended in 10 mM 3-(N-morpholino)propanesulphonic acid buffer (MOPS)/0.9% NaCL solution pH 7.4. The suspension is diluted to one hundredth of the original blood volume. In each case, 99 xcexcl of the suspension are admixed with 10 xcexcl of a suitable concentration of the substance to be investigated, and the mixture is incubated at 30xc2x0 C. for 5 min. 5 xcexcl of a 4 mM adenosine solution are then added, and the mixture is incubated at 30xc2x0 C. for another 15 min. The samples are then centrifuged at 3000 g for 5 min and in each case 700 xcexcl of the supernatant are admixed with 28 xcexcl of 70% strength HClO4, allowed to stand in an ice bath for 30 min and centrifuged at 16,000 g for 3 min, and 350 xcexcl of the sample are neutralized using 30 xcexcl of 5N NaOH. 50 xcexcl of the sample are applied to a column (Waters Symmetry C18 5 xcexcm 3.9xc3x97150 mm). A Spherisorb ODS II 5 xcexcm 4.6xc3x9710 mm column is used as precolumn. The mobile phase used is a gradient of 50 mM KH2PO4/5 mM tributylamine pH 7 (mobile phase A) and a mixture of mobile phase A/methanol 1/1 (mobile phase B). The gradient is from 10 to 40% B, at a flow rate of 0.5 ml/min. The adenosine which is present is quantified by its absorption at 260 nm, as are the hypoxanthine and inosine formed. The IC50 is the concentration of active compound at which, 15 min after addition of adenosine, 50% of the adenosine concentration originally employed is still present.
Using this test, the IC50 value determined for Example 1-1 was 30 nM, that for Example 1-3 was 20 nM, that for Example 1-14 was 30 nM, that for Example 1-33 was 40 nM, that for Example 2-1 was 20 nM and that for Example 2-18 was 20 nM.
3. In Vivo Test Model for Testing Adenosine-Uptake Inhibitors
Adult FBI (Foxhound-Beagle-Irish-Setter) dogs (20-30 kg) are initially anaesthetized using a combination of trapanal 500 mg and alloferin 55 mg. Anaesthesia is maintained by infusion of a mixture of fentanyl 0.072 mg/kg, alloferin 0.02 mg/kg and dihydrobenzpyridyl 0.25 mg/kgxc3x97min. The animals are intubated and ventilated with a mixture of O2/N2O ⅕ using an Engstrxc3x6m ventilation pump at 16 breaths per min and a volume of 18-24 ml/kg. The body temperature is maintained at 38xc2x0 C.xc2x10.1xc2x0 C. Arterial blood pressure is measured via a catheter in the femoral artery. Thoractomy is carried out on the left side at the fifth intercostal space. The lung is pushed back and fixed and a cut is made in the pericardium. A proximal section of the LAD distally to the first diagonal branch is exposed and a calibrated electromagnetic flow sensor (Gould Statham, model SP7515) is placed around the vessel and attached to a flow meter (Statham, model SP-2202). Distally to the flow sensor, a mechanical occluder is attached such that there are no branches in between flow sensor and occluder.
Using a catheter in the femoral vein, blood samples are taken and substances administered. A peripheral ECG is recorded using needles which are fixed subcutaneously. A microtip pressure manometer (Millar model PC-350) is pushed through the left atrium to measure the pressure in the left ventricle. Measurement of the heart frequency is triggered by the R wave of the ECG. During the entire experiment, the haemodynamic parameters and coronary flow are recorded using a multi-event recorder.
A four-minute occlusion causes reactive hyperaemia. The difference between the coronary flow under control conditions and the maximum flow during the reactive hyperaemia is measured. The time which is required to achieve half of this maximum flow in the drop is a suitable parameter to assess the reactive hyperaemia.
After a stabilization period of one hour, the experiment is started with a four-minute occlusion. Thirty minutes later, the substance is administered (i.v.) which is, after two minutes, followed by re-occlusion. The reactive hyperaemia after verum and placebo is compared.
4. Measurement of the Plasma Concentration of Adenosine-Uptake Inhibitors Following Oral Administration to Mice
Test principle:
Following oral administration, blood samples are taken from the mice and the concentration of the active compound in the blood is measured by the functional inhibition of the adenosine uptake in rabbit erythrocytes.
The substances were administered in a dosage of 10 mg/kg and an administration volume of 10 ml/kg using a stomach tube. The solvent used was polyethylene glycol 400/ethanol 9:1. After one hour, the animals were anaesthetized and, by puncture of the heart, about 0.5 to 0.7 ml of blood were taken. The blood was precipitated in 5 times its volume of acetonitrile, kept in an ice bath for 30 minutes and then centrifuged at 16,000 g in an Eppendorf centrifuge for 5 minutes. At room temperature, the supernatant was evaporated to dryness in a Speedvac. The dried samples were initially wetted with 20 xcexcl of DMSO and then admixed with 1 ml of 10 mM of 3-(N-morpholino)propanesulphonic acid buffer (MOPS)/0.9% aqueous sodium chloride solution pH 7.4 and kept in an ultrasonic bath for 15 minutes. They were then centrifuged at 16,000 g for 5 minutes.
In each case, 500 xcexcl of extract; 200 xcexcl of extract and 300 xcexcl of the abovementioned buffer; 100 xcexcl of extract and 400 xcexcl of buffer; 50 xcexcl of extract and 450 xcexcl of buffer were mixed with a suspension of in each case 500 xcexcl of rabbit erythrocytes. [The erythrocytes were isolated as described under Experiment 2 (xe2x80x9cInhibition of the adenosine uptake in rabbit erythrocytesxe2x80x9d) and diluted to fifty times the original blood volume]. As described under Experiment 2, adenosine was added after 5 minutes and the adenosine uptake was measured. The inhibition of adenosine uptake can be used to calculate the concentration of the inhibitor in the sample, since the inhibitory effect of the adenosine-uptake inhibitor was determined beforehand by a concentration curve using the method described in Experiment 2.
5. Mouse Angiogenesis Model
To test the effect of the adenosine-uptake inhibitors on collateralization and neovascularization, a mouse model for angiogenesis was developed. To this end, a femoral artery of the mouse is ligated at the upper end of the thigh. This induces chronic ischaemia of the hind leg in question. The other hind leg serves as individual control. To exclude residual flow through the ligated vessel, two ligatures are applied, and the vessel is cut in between. A few days after this operation, the treatment is started.
As a measurement parameter during the ongoing experiment, the temperatures of the paws of the two hind legs are measured. Owing to poorer circulation, the ischaemic hind leg has a lower absolute temperature. In each case, the temperature difference between the paws of the hind legs is calculated. This individual temperature difference is determined in various treatment groups as a function of the dose and in comparison with an untreated control. In this model, adenosine-uptake inhibitors significantly improve the circulation of the ischaemic hind leg in comparison with the corresponding controls.
The novel active compounds can be converted in a known manner into the customary formulations, such as tablets, sugar-coated tablets, pills, granules, aerosols, syrups, emulsions, suspensions and solutions. In this connection, the therapeutically active compound should in each case be present in a concentration of approximately 0.5 to 90% by weight of the total mixture, i.e. in amounts which are sufficient in order to achieve the dosage range indicated. In addition to the active compounds of the formula (I), the formulations may also comprise other pharmaceutically active compounds.
The formulations are prepared, for example, by extending the active compounds with inert non-toxic pharmaceutically suitable auxiliaries. Auxiliaries which may be mentioned are, for example: water, non-toxic organic solvents, such as, for example, paraffins, vegetable oils (for example sesame oil), alcohols (for example ethanol, glycerol), glycols (for example polyethylene glycol), solid carriers, such as natural or synthetic ground minerals (for example talc or silicates), sugar (for example lactose), emulsifiers, dispersants (for example polyvinylpyrrolidone) and glidants (for example magnesium sulphate).
Administration is carried out in a customary manner, preferably orally, transdermally, parenterally, perlingually, intravenously; particularly preferably orally or intravenously.
In general, it has proven advantageous in the case of intravenous administration to administer amounts of approximately 0.0001 to 10 mg/kg, preferably approximately 0.003 to 1 mg/kg, of body weight, to achieve effective results. In the case of oral administration, 0.1 to 20 mg/kg, preferably 0.3 to 10 mg/kg, of body weight are employed.
In spite of this, if appropriate, it may be necessary to depart from the amounts mentioned, namely depending on the body weight or on the type of administration route, on the individual response towards the medicament, the manner of its formulation and the time or interval at which administration takes place. Thus, in some cases it may be adequate to manage with less than the abovementioned minimum amount, while in other cases the upper limit mentioned has to be exceeded. It may be advisable to divide this amount into a number of individual doses over the course of the day or to have a delayed release of active compound from the formulation over a relatively long period of time.
Below, the present invention is illustrated using the following preferred examples; however, these examples do not limit the invention in any way.
Unless indicated otherwise, all amounts are in percent by weight; in the case of solvent mixtures, ratios by volume are given.